Applicant: I am a cardiologist who plans to pursue an academic career that centers on thrombosis in cardiovascular disease. This goal is the result of the basic and clinical experience I acquired as a research fellow in the Hematology Research Unit at Mayo Clinic and my exposure to many patients with thrombotic disease during my clinical cardiology training. At present I am an instructor within the Division of Cardiology at the University of Michigan. Greater than 80% of my time is spent in basic research. Research: The broad objective of this application is to understand how endothelial cell-derived plasminogen activator inhibitor, or PAI-1, functions to regulate fibrinolysis and how it participates in human disease. PAI-1 is considered the primary physiologic inhibitor of t-PA and urokinase, and several lines of evidence implicate PAI-1 in the pathogenesis of thrombotic and hemorrhagic disorders. The specific aim of the proposed studies is to define functional domains in PAI-1. Other than the localization of the reactive center to the carboxyl portion of the inhibitor, little is known about the structural determinants of the interaction of PAI-1 with plasminogen activators or regulatory molecules. To address this issue, a series of mutations within the mature PAI-1 cDNA will be generated. These mutants will be expressed in E. coli and functionally characterized. PAI-1 mutants will also be used to map epitopes of a panel of anti-PAI-1 monoclonal antibodies, and the effects of these antibodies on PAI-1 function will be determined. In separate experiments, 2 patients with PAI-1 deficiency and abnormal bleeding will be studied to determine if a defect is present within their PAI-1 gene. Environment: Dr. David Ginsburg will serve as sponsor for the proposed studies. Dr. Ginsburg has a long-standing interest in the basic mechanisms of coagulation and fibrinolysis. His laboratory offers me ready access to the equipment and personnel necessary to obtain an in-depth exposure to the methods of molecular biology and their application to human disease. In addition, the University of Michigan is actively involved in clinical research related to thrombosis, most notably the use of thrombolytic therapy in patients with myocardial infarction. Upon completion of training with Dr. Ginsburg, I hope to apply basic techniques to these studies. Hence, I consider this a superb environment in which to prepare for a career as an independent investigator capable of combining basic laboratory research with clinical cardiology.